JPH0477435A - Cancer metastasis suppressing agent - Google Patents

Abstract

PURPOSE:To provide a cancer metastasis suppressing agent containing a polypeptide corresponding to a heparin-bonding polypeptide in fibronectin molecule and free from blood platelet coagulation inhibiting action. CONSTITUTION:The objective agent contains, as an active component, a polypeptide included in the heparin-bonding domain of fibronectin and having heparin- bonding activity, e.g. a polypeptide composed of 271 amino acid residues (Ala<1690>-Thr<1960>) and having the amino acid sequence of formula. The daily dose of the active component compound is 1-100mg/kg for parenteral administration and 5-500mg/kg for oral administration. The above polypeptide can be produced by culturing an E.coli [E.coli HB 101/pHD 101 (FERM BP-2264)] containing introduced plasmid capable of expressing the polypeptide composed of 271 amino acid residues.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a cancer metastasis inhibitor, and more particularly to the use of a functional domain in the fibronectin molecule, which is an oily polypeptide in living organisms, as a cancer metastasis inhibitor. .

[Conventional technology]

Various drug treatments and surgical treatments have been used to treat cancer, but their effects cannot be said to be sufficient.

In particular, postoperative recurrence in surgical therapy has become a serious problem. Cancer recurrence is caused by cancer metastasis, which has led to the development of cancer metastasis inhibitors.

It is known that the platelet aggregation activity of cancer cells themselves is involved in cancer metastasis, and the platelet aggregation inhibitor ticlopidine is known to have an effect of suppressing cancer metastasis.

[Problem to be solved by the invention]

However, platelet aggregation inhibitors have an adverse effect because they have the effect of inhibiting not only cancer cells but also platelet aggregation in general.

An object of the present invention is to provide a new cancer metastasis inhibitor that does not inhibit platelet aggregation.

[Failure to solve the problem]

To summarize the present invention, the present invention relates to a cancer metastasis inhibitor,
It is characterized by containing a polypeptide corresponding to the heparin-binding polypeptide in the fibronectin molecule.

The present inventors conducted intensive research on the functional domain of fibronectin (hereinafter abbreviated as FN), which is an in vivo substance, and found that heparin-conjugated polypeptide has a remarkable effect of inhibiting cancer metastasis and inhibits platelet aggregation. They discovered that this does not occur, and completed the present invention.

The present invention will be explained in detail below.

In FN, a polypeptide with a molecular weight of about 250,000 forms a dimer with an S-8 bond near the C-terminus.

The intramolecular amino acid sequence has a repeating structure and consists of ■, ■, ■
Divided into types. Furthermore, it has a domain structure with various functions, and exhibits cell adhesion, binding activity to collagen, heparin, fibrin, etc.

As a result of research into new uses of the functional domain of FN, the present inventors found that heparin-binding polypeptides have a significant cancer metastasis suppressive effect.

In the present invention, the heparin-binding polypeptide is not particularly limited as long as it is a polypeptide corresponding to a polypeptide that is contained in the heparin-binding domain of FN and has heparin-binding activity.

Examples of the Kamikita polypeptide include the heparin-binding polypeptide disclosed in Japanese Patent Application No. 1-131453 created by the present inventors. circle.

An example of a heparin-binding polypeptide will be described below.

271 amino acid residue polypeptide 7 hr l 960) has the following formula I: Human! a'lle Pr. AIa pr. Thr
AspThr Gln Vat Thr Pro T
hr 5erGin Trp Thr Pro Pro
^sn ValGly Tyr Ar-g Val A
rg Val ThrLys Thr Gly Pro
Met Lys GluAla Pro Asp S
er Ser Ser Ser ValGly Leu M
et Val Ala Thr LysSer
Val Tyr Ala Leu Lys AspS
er Arg Pro Ala Gin Gly Va
lLeu Glu Asn Val Ser Pro
Pr.

(Ala1690 Seu Seu 1n Pr.

1e Val Tyr Thr Val 8rg Lys Ser Leu Lys Eight sns Val Glu Leu Thr 8rg Phe 81a Thr Glu Seu Ser νa) Thr Thr AIa Arg Val Thr　l1e lie Thr Ala　Asn 1ie Lys Thr　Gly lie Tyr Arg 5er Thr　Ala Phe Leu Vat 5er Thr　Gly Gly 5er Pro Arg Thr　Gly lie Tyr Lys Ser Thr Thr Ser Gly Ty Pr.

Leu Seu Ser lie AIa rp Tyr Pr.

Pr.

Leu Val Glu Asp rp Phe G1 mouth Asp Gin Tyr Pr.

Asp Thr Gin 1e Pr.

1y Glu 1e Pr.

AIa Arg Gin Thr Val Pr.

Thr Val AIa Thr Pr.

1e Arg Val Pr.

AIa Leu Thr Thr Val Pr.

Arg Gly Leu Val Pr.

Pr.

Pr.

Lys Glu Thr Gly Leu lie Glu Lys Asp lie Ser Thr Asn lie Ser Asn Arg Tyr Val Glu Thr Lys Gly Thr Thr AIa Gin Tyr Asp Asp Asp Asn Ser AIa Glu Val AIa Glu Asn Arg Thr Glu Val Arg Thr Tyr Asn AIa Leu Leu Arg Lys Pr.

Thr Tyr Asn Yes 1e Thr P'r.

Thr lie Lys AIa Ser Ar)H Leu 1e Pr.

Arg lie Thr Gin Yes [I) It is characterized by being represented by the amino acid sequence represented by

In addition, in this specification, page numbers assigned to amino acids are from ENBL Data Bank (EMBL DATA BAN).
The number of amino acid residues from the N-terminus assigned to the amino acids obtained by translation of the FN cDNA sequence of K) is shown.

The manufacturing method is shown in Japanese Patent Application No. 1-131453, and will be specifically explained below.

cONA encoding the heparin-binding domain of human FN
Plasmid pLF 2435 containing the fragment is described in Biochemistry, Vol. 25, pp. 4936-4941 (1986).
This is a plasmid reconstructed by ligating the coding regions of pLF 2.4.3 and 5.

Excise the necessary cDNA fragment from this plasmid pLF 2435 with a restriction enzyme, and create a synthetic DNA containing a start codon on the 5' side and a stop codon on the 3' side. After ligating with J gauze, the two heparin-binding domains are ligated into an appropriate expression vector.
71 amino acid residue polypeptide (A1a+5so-Th
plasmid expressing r+5so) p)l[l 101
is constructed.

All existing expression vectors can be used, but for example, p[Jc 118N/pUC11
9N [FBBS Letters
Good results can be obtained by using s) Vol. 223, pp. 174-180 (1987) and its derivatives. By introducing this plasmid into E. coli, the heparin-binding polypeptide can be expressed and its properties can be investigated.

271 amino acid residue polypeptide (Ala1690Th
Escherichia coli Bsc into which a plasmid expressing
herichia coli He 101/pH01
01 is FIliRM BP-
2264).

Purification of this heparin-binding polypeptide from recombinant
For example, do as follows. After culturing the recombinant E. coli in a medium such as L (iiD gloss) and collecting the bacteria, a bacterial cell suspension is obtained by ultrasonication, and this is centrifuged to obtain a supernatant. After dialysis of the supernatant, D8AIE It is passed through an ion exchanger column, and then subjected to affinity chromatography using a CM ion exchanger and/or heparin-agarose, etc. Through the above operations, the polypeptide of interest can be purified.

When the polypeptide of the present invention obtained as described above is used as a medicine, it may be formulated by a conventional method together with a pharmaceutical carrier if necessary, and administered orally or parenterally. A pharmacologically acceptable excipient or carrier is selected, and its type and composition vary depending on the route and method of administration. For example, water, alcohols, animal or vegetable oils such as soybean oil, olive oil, mineral oil, or synthetic oils are used as liquid carriers.

As solid carriers, sugars such as maltose and sucrose, amino acids, cellulose derivatives such as hydroxypropyl cellulose, organic acid salts such as magnesium stearate, etc. are used.

In the case of injections, the dissolving solution preferably includes physiological saline, various buffer solutions, saccharide solutions such as glucose, inositol, manganese, nidol, and lactose, and glycols such as ethylene glycol and polyethylene glycol. In addition, sugars such as inositol, mannitol, lactose, and sucrose,
A lyophilized preparation is prepared together with excipients such as amino acids such as phenylalanine, and at the time of administration, it is dissolved in a suitable solvent for injection, such as a liquid for intravenous administration such as sterile water, physiological saline, glucose solution, electrolyte solution, amino acid solution, etc. It can also be administered. The content of the polypeptide of the present invention in a preparation varies depending on the preparation, but is usually 0.1 to 100% by weight, preferably 1 to 98% by weight. For example, in the case of injections,
It is generally desirable to contain the active ingredient in an amount of 0.1 to 30% by weight, preferably 1 to 10% by weight. When administered orally, it is used in the form of tablets, capsules, powders, granules, liquids, dry syrups, etc. together with the solid carrier or liquid carrier. Capsules, granules and powders generally contain from 5 to 100% by weight of active ingredient, preferably from 25 to 98%.

The dose is determined depending on the patient's age, weight, symptoms, therapeutic purpose, etc., but the therapeutic dose is generally 1 to 100 mg for parenteral administration.
mg/kg/day, 5-500■/kg by oral administration
/ day.

[Effect]

Next, the physiological activity of the polypeptide of the present invention will be illustrated through experimental examples.

Experimental Example 1 Cancer Metastasis Suppressing Effect CDF, L5178Y intravenously administered to mice (5 mice per group)
- ML25 leukemia 11 cells 4xl (1' cells, 2 of each concentration)
It is mixed with a 71 amino acid residue polypeptide and transplanted. In addition, only leukemia cells are transplanted as a control.

14 days after leukemia cell transplantation, the mice in each lipstick group are sacrificed, the liver and pancreas are removed, and their weights are compared. Table 1 shows the results.
Shown below.

Table 500 1.42±OJ5 0.11±0.02 As shown above, the metastasis of leukemia cells to the liver and pancreas was inhibited in the group administered with the polypeptide of the present invention. Concentration 5 μM ADP and 1
00 μg/- of collagen). Human platelets (5
xl O5/μA) Final concentration 100μg in 0.25m
/rrLl of the polypeptide of the present invention was added, and 30 seconds later, ADP or collagen was added.

In the polypeptide of the present invention, no inhibitory effect on platelet aggregation caused by ADP and collagen was observed.

Experimental Example 3 Acute Toxicity Test The polypeptide of the present invention was administered intravenously to CDF and mice. No toxicity was observed at 100 μ/kg administration.

As shown in the above experimental examples, although the polypeptide of the present invention has no effect of inhibiting platelet aggregation, L51
It shows a significant metastasis prevention effect in a metastasis model system using 78Y leukemia cells, and is effective against gastric cancer, lung cancer, colorectal cancer, breast cancer,
It is a useful substance that effectively prevents metastasis of cancer cells such as prostate cancer, cervical cancer, and kidney cancer.

〔Example〕

Next, the present invention will be specifically explained with reference to Examples, but the scope of the present invention is not limited to the Examples.

In addition, on each side, parts mean parts by weight.

Formulation Example 1 PBS was added to 30 parts of a 271 amino acid residue polypeptide to make a total amount of 2000 parts, and the solution was dissolved, followed by sterilization filtration using a Millipore filter - GS type. 2 g of this filtrate was placed in a 10-bar vial and lyophilized to obtain a lyophilized injection containing 30 mg of the polypeptide per vial.

Formulation Example 2 50 parts of a 271 amino acid residue polypeptide, 600 parts of lactose, 330 parts of crystalline cellulose and 20 parts of hydroxypropyl cellulose were thoroughly mixed, compressed using a roll compactor, and crushed to give 16 The mixture was sieved to obtain a particle size between 60 mesh and granules.

〔Effect of the invention〕

The polypeptide of the present invention is a biologically relevant substance, has high safety, and is useful as a cancer metastasis inhibitor. It also has a remarkable effect in that it can be supplied in large quantities through genetic engineering.

Procedural amendment (voluntary) August 2, 1990 Toshi Uematsu, Commissioner of the Japanese Patent Office 1, Indication of the case 1990 Patent Application No. 187137 2
, Title of the invention Cancer metastasis inhibitor 3, Relationship to the amended person's case Patent applicant address 609 Takenaka-cho, Fushimi-ku, Kyoto-shi, Kyoto Name
Representative of Hinshuzo Co., Ltd. 1) Tetsu Be 5, date of amendment order 6, subject of amendment voluntary amendment 7, and the detailed description of the invention column in the statement of contents of the amendment are amended as follows.

(1) "rL (in) Pros" in the bottom three lines of page 7 of the specification is corrected to "L-Bros".

Claims (1)

[Claims] 1. A cancer metastasis inhibitor characterized by containing a polypeptide corresponding to the heparin-binding polypeptide in the fibronectin molecule.